U.S. patent number 10,597,291 [Application Number 15/796,646] was granted by the patent office on 2020-03-24 for disposable microfluidic cartridge.
This patent grant is currently assigned to The University of British Columbia. The grantee listed for this patent is The University of British Columbia. Invention is credited to Aysha Ansari, Gesine Heuck, Timothy Leaver, Kevin Ou, Euan Ramsay, Robert James Taylor, Anitha Thomas, Colin Walsh, Andre Wild.
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United States Patent |
10,597,291 |
Wild , et al. |
March 24, 2020 |
Disposable microfluidic cartridge
Abstract
The present disclosure is directed towards a disposable
microfluidic cartridge configured for use in a system for the small
scale production of nanoparticles used in scientific research or
therapeutic applications. The system can be used to produce a wide
variety of nanoparticles, including but not limited to lipid and
polymer nanoparticles, carrying a variety of payloads. The system
provides for a simple workflow which in certain embodiments can be
used to produce a sterile product.
Inventors: |
Wild; Andre (Vancouver,
CA), Leaver; Timothy (Delta, CA), Walsh;
Colin (Belmont, CA), Heuck; Gesine (Vancouver,
CA), Thomas; Anitha (New Westminster, CA),
Ansari; Aysha (Calgary, CA), Ou; Kevin (Toronto,
CA), Taylor; Robert James (Vancouver, CA),
Ramsay; Euan (Vancouver, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
The University of British Columbia |
Vancouver |
N/A |
CA |
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Assignee: |
The University of British
Columbia (Vancouver, CA)
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Family
ID: |
57199399 |
Appl.
No.: |
15/796,646 |
Filed: |
October 27, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20180111830 A1 |
Apr 26, 2018 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/US2016/029879 |
Apr 28, 2016 |
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62275630 |
Jan 6, 2016 |
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62154043 |
Apr 28, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01F
5/0644 (20130101); B01L 9/527 (20130101); B01F
13/0064 (20130101); B82Y 40/00 (20130101); B01F
13/0059 (20130101); B81B 1/00 (20130101); B01F
5/0647 (20130101); B01F 5/061 (20130101); B01F
2005/0636 (20130101); B01L 2300/0816 (20130101); B01L
2200/025 (20130101) |
Current International
Class: |
B01F
13/00 (20060101); B01L 9/00 (20060101); B01F
5/06 (20060101); B82Y 40/00 (20110101); B81B
1/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2 918 368 |
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Jan 2015 |
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CA |
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2 927 358 |
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Apr 2015 |
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CA |
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103906503 |
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Jul 2014 |
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CN |
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103 56 308 |
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Jun 2005 |
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DE |
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2008/039209 |
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Apr 2008 |
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WO |
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2014/172045 |
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Oct 2014 |
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WO |
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Other References
Howell, P.B. Jr., et al., "Design and Evaluation of a Dean
Vortex-Based Micromixer," Lab on a Chip 4(6):663-669, Dec. 2004.
(Year: 2004). cited by examiner .
Deshpande, A., "Polystyrene Properties," Buzzle, Feb. 1, 2013,
<http://www.buzzle.com/articles/polystyreneproperties.html>
[retrieved Jul. 11, 2016], pp. 1-6; especially p. 3, table entitled
"Mechanical Properties." cited by applicant .
Howell, P.B. Jr., et al., "Design and Evaluation of a Dean
Vortex-Based Micromixer," Lab on a Chip 4(6):663-669, Dec. 2004.
cited by applicant .
International Search Report and Written Opinion dated Sep. 2, 2016,
issued in corresponding International Application No.
PCT/US2016/29879, filed Apr. 28, 2016, 18 pages. cited by applicant
.
Partial Search Report and Invitation to Pay Additional Fees dated
Sep. 21, 2018, issued in corresponding European Application No.
16756307.1, filed Feb. 24, 2016, 23 pages. cited by applicant .
International Preliminary Report on Patentability dated Nov. 9,
2017, issued in corresponding International Application No.
PCT/US2016/029879, filed Apr. 28, 2016, 15 pages. cited by
applicant .
Extended European Search Report dated Feb. 8, 2019, issued in
corresponding European Application No. 16756307.1, filed Feb. 24,
2016, 17 pages. cited by applicant .
Supplementary European Search Report dated Aug. 1, 2019, issued in
corresponding European Application No. 16882817.6, filed Aug. 24,
2016, 9 pages. cited by applicant .
Chen et al., "Optimal Designs of Staggered Dean Vortex
Micromixers," International Journal of Molecular Sciences
12(6):3500-3524, Jan. 11, 2011. cited by applicant .
First Office Action dated Jun. 24, 2019, issued in corresponding
Chinese Application No. 201680022904.1, filed Feb. 24, 2016, 6
pages. cited by applicant .
Search Report dated Jun. 24, 2019, issued in corresponding Chinese
Application No. 201680022904.1, filed Feb. 24, 2016, 3 pages. cited
by applicant .
Extended European Search Report dated Dec. 14, 2018, issued in
corresponding European Application No. 16787188.8, filed Apr. 28,
2016, 9 pages. cited by applicant .
International Search Report and Written Opinion dated May 17, 2016,
issued in corresponding International Application No.
PCT/US16/19414, filed Feb. 24, 2016, 20 pages. cited by applicant
.
International Search Report and Written Opinion dated Nov. 2, 2016,
issued in corresponding International Application No.
PCT/CA2016/050997, filed Aug. 24, 2016, 6 pages. cited by applicant
.
International Preliminary Report on Patentability dated Aug. 29,
2017, issued in corresponding International Application No.
PCT/US2016/019414, filed Feb. 24, 2016, 12 pages. cited by
applicant.
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Primary Examiner: Wecker; Jennifer
Attorney, Agent or Firm: Christensen O'Connor Johnson
Kindness PLLC
Parent Case Text
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation of International Application No.
PCT/US2016/029879, filed Apr. 28, 2016, which claims the benefit of
U.S. Patent Application No. 62/154,043, filed Apr. 28, 2015, and
U.S. Patent Application No. 62/275,630, filed Jan. 6, 2016, the
disclosures of which are hereby incorporated by reference in their
entirety.
Claims
The embodiments of the invention in which an exclusive property or
privilege is claimed are defined as follows:
1. A microfluidic cartridge that is disposable and is configured to
mix a first solution with a second solution to produce a mixed
solution, the microfluidic cartridge comprising: (A) a carrier,
comprising a first inlet connector, a second inlet connector, and
an outlet opening; (B) a microfluidic structure integrally
incorporated into the carrier, the microfluidic structure
comprising: (I) a first inlet microchannel configured to receive
fluid from first inlet connector; (II) a second inlet microchannel
configured to receive fluid from the second inlet connector; (III)
a mixer configured to: receive a first solution from the first
inlet microchannel and a second solution from the second inlet
microchannel; mix the first solution and the second solution to
provide a mixed solution, and direct the mixed solution into an
outlet microchannel; and (IV) the outlet microchannel, which is in
fluid communication with the outlet opening, wherein the mixer is a
Dean Vortex Bifurcating Mixer (DVBM) comprising an inlet leading to
a first leg channel and a second leg channel that define a
circumference of a first toroidal mixing element.
2. The microfluidic cartridge of claim 1, wherein the microfluidic
cartridge is configured for a single use.
3. The microfluidic cartridge of claim 1, further comprising a
securing mechanism configured to secure the microfluidic cartridge
to a holder.
4. The microfluidic cartridge of claim 3, wherein the securing
mechanism comprises one or more magnets.
5. The microfluidic cartridge of claim 3, wherein the securing
mechanism comprises a carrier locking feature on the carrier that
is configured to lock with a holder locking feature on a compatible
holder for the microfluidic cartridge.
6. The microfluidic cartridge of claim 1, wherein the carrier is at
least 90%, by weight, polymer.
7. The microfluidic cartridge of claim 1, wherein the carrier does
not include metal on an exterior surface.
8. The microfluidic cartridge of claim 1, wherein the microfluidic
structure is not separable from the carrier.
9. The microfluidic cartridge of claim 1, wherein the carrier
encloses the microfluidic structure.
10. The microfluidic cartridge of claim 1, wherein the carrier
comprises a first portion, comprising the first inlet connector,
the second inlet connector, and the outlet opening and a second
portion, wherein the first portion and the second portion join to
seal the microfluidic structure between the first portion and the
second portion.
11. The microfluidic cartridge of claim 10, wherein the first
portion and the second portion join to enclose the microfluidic
structure.
12. The microfluidic cartridge of claim 10, wherein the first
portion is at least 90%, by weight, polymer.
13. The microfluidic cartridge of claim 10, wherein the first
portion or the second portion comprises the microfluidic
structure.
14. The microfluidic cartridge of claim 1, wherein the first leg
channel of the DVBM has a first impedance and the second leg
channel of the DVBM has a second impedance, the first impedance
being greater than the second impedance.
15. The microfluidic cartridge of claim 14, wherein the DVBM
comprises a third leg channel and a fourth leg channel that define
a circumference of a second toroidal mixing element that is in
fluid communication with the first toroidal mixing element via a
neck region, the third leg channel having a third impedance and the
fourth leg channel having a fourth impedance, the third impedance
being greater than the fourth impedance.
16. The microfluidic cartridge of claim 15, wherein a ratio of the
first impedance to the second impedance is about equal to a ratio
of the third impedance to the fourth impedance.
17. The microfluidic cartridge of claim 1, wherein the DVBM
comprises a third leg channel and a fourth leg channel that define
a circumference of a second toroidal mixing element that is in
fluid communication with the first toroidal mixing element via a
neck region, the first leg channel having a first length, the
second leg channel having a second length, the third leg channel
having a third length, the fourth leg channel having a fourth
length, the first length being greater than the second length and
the third length being greater than the fourth length.
18. The microfluidic cartridge of claim 1, wherein the first leg
channel and the second leg channel each have a hydrodynamic
diameter of about 20 microns to about 2 mm.
19. The microfluidic cartridge of claim 1, wherein the mixer is
sized and configured to mix the first solution and the second
solution at a Reynolds number of less than 1000.
20. The microfluidic cartridge of claim 1, wherein the mixer
comprises between two and twenty toroidal mixing elements arranged
in series.
21. The microfluidic cartridge of claim 1, wherein the microfluidic
cartridge is sterile.
22. The microfluidic cartridge of claim 1, wherein the microfluidic
cartridge includes a sterile fluid path, from the first inlet
connector and the second inlet connector, through the microfluidic
structure, and to the outlet opening.
23. A sterile package filled with sterile contents, comprising a
microfluidic cartridge of claim 1 in a sterile state and sealed
within the sterile package.
24. A method of forming nanoparticles, comprising flowing a first
solution and a second solution through a microfluidic cartridge
according to claim 1 and forming a nanoparticle solution in the
mixer.
Description
BACKGROUND
Microfluidic mixers have found use in research labs as a means for
mixing small volumes of fluids in order to conserve precious
materials and/or prepare small (e.g., single batch) amounts of
product. Synthesis of lipid nanoparticles is one of many uses for
microfluidic mixers.
As microfluidics finds an expanded role in research, development,
and production, users will desire lower costs, easier
manufacturing, and simplified operation of microfluidic mixers
while maximizing performance.
SUMMARY
This summary is provided to introduce a selection of concepts in a
simplified form that are further described below in the Detailed
Description. This summary is not intended to identify key features
of the claimed subject matter, nor is it intended to be used as an
aid in determining the scope of the claimed subject matter.
In one aspect, a microfluidic cartridge is provided that is
disposable and is configured to mix a first solution with a second
solution to produce a mixed solution. In one embodiment, the
microfluidic cartridge includes:
(A) a carrier, comprising a first inlet connector, a second inlet
connector, and an outlet opening;
(B) a first microfluidic structure integrally incorporated into the
carrier, the microfluidic structure comprising: (I) a first inlet
microchannel configured to receive fluid from first inlet
connector;
(II) a second inlet microchannel configured to receive fluid from
the second inlet connector;
(III) a first mixer configured to:
receive a first solution from the first inlet microchannel and a
second solution from the second inlet microchannel;
mix the first solution and the second solution to provide a mixed
solution, and
direct the mixed solution into an outlet microchannel; and
(IV) the outlet microchannel, which is in fluid communication with
the outlet opening.
In another aspect, a sterile package filled with sterile contents
is provided. In one embodiment, the sterile package includes a
microfluidic cartridge according to any of the embodiments
disclosed herein in a sterile state and sealed within the sterile
package.
In another aspect, a method of forming nanoparticles is provided.
In one embodiment, the method includes flowing a first solution and
a second solution through a microfluidic cartridge according to any
of the disclosed embodiments and forming a nanoparticle solution in
the first mixer.
DESCRIPTION OF THE DRAWINGS
The foregoing aspects and many of the attendant advantages of this
invention will become more readily appreciated as the same become
better understood by reference to the following detailed
description, when taken in conjunction with the accompanying
drawings, wherein:
FIGS. 1A-1C illustrate microfluidic cartridges in accordance with
embodiments disclosed herein;
FIGS. 2 and 3 are exploded views of the microfluidic cartridge of
FIGS. 1A and 1B;
FIG. 4A is a schematic illustration of a staggered herringbone
mixer of the type incorporated in a microfluidic cartridge in
accordance with certain embodiments disclosed herein;
FIG. 4B is a schematic illustration of a toroidal pair Dean vortex
bifurcating mixers (DVBM) in accordance with the disclosed
embodiments. FIG. 4C is a photograph of an exemplary toroidal DVBM
mixer, mixing two solutions, in accordance with the disclosed
embodiments.
FIG. 5A illustrates a microfluidic cartridge connected to solution
reservoirs (syringes) with the aid of a holder, in accordance with
embodiments disclosed herein; and
FIGS. 5B-5H are photographs depicting exemplary microfluidic
cartridges in accordance with embodiments disclosed herein.
DETAILED DESCRIPTION
The present disclosure is directed towards a disposable
microfluidic cartridge configured for use in a system for the small
scale production of nanoparticles used in scientific research or
therapeutic applications. The system can be used to produce a wide
variety of nanoparticles, including but not limited to lipid and
polymer nanoparticles, carrying a variety of payloads. The system
provides for a simple workflow which in certain embodiments can be
used to produce a sterile product.
The microfluidic cartridge provides a convenient, disposable
platform for combining two or more microfluidic streams within a
microfluidic mixer. Currently produced microfluidic mixer systems
include a permanent (non-disposable, reusable) metal housing into
which a microfluidic "chip" is placed (e.g., as used in the
NanoAssemblr manufactured by Precision Nanosystems Inc. of
Vancouver, BC). The metal housing includes the inlets that connect
to sources of solution (e.g., pumps or syringes). For each use, the
microfluidic layer must be carefully positioned within the metal
housing and sealed against the inlets and outlet in order to
produce a fluid-tight path through the microfluidics and the metal
housing. Other microfluidic products include non-disposable,
non-metal mixers, such as those sold by Dolomite (Royston, UK). The
disclosed microfluidic cartridge provides a benefit over these
two-part systems that require laborious set-up time--in the form of
fitting the microfluidic layer into the metal holder. The disclosed
embodiments also allow for simplified access to sterile
microfluidic mixing due to the ability to provide a pre-sterilized
microfluidic cartridge having a sterile fluidic path.
Furthermore, the described system minimizes user assembly by
integrating fittings and microfluidics into one cartridge. This
integration allows for more reliable operation (eliminating user
assembly steps), higher operating pressures and minimizes internal
volume. The disposable nature of the cartridge reduces the risk of
cross-contamination and reduces experimental time by eliminating
the need for washing.
In particular, the present disclosure provides an apparatus for the
manufacture of nanoparticles, which enables the simple, rapid and
reproducible manufacture of nanoparticles at laboratory scales
(0.5-20 mL) for applications such as fundamental research, particle
characterization, material screening and in-vitro and in-vivo
studies using a disposable cartridge. This disclosure employs
microfluidics which has the advantage of exquisite control over
environmental factors during manufacture, and microfluidics
possesses the further advantage of allowing seamless scale-up via
parallelization. The disclosed embodiments are configured to mix
the first solution with the second solution through a microfluidic
mixer. Many methods for this mixing process are known. Compatible
microfluidic mixing methods and devices are disclosed in:
(1) U.S. patent application Ser. No. 13/464,690, which is a
continuation of PCT/CA2010/001766, filed Nov. 4, 2010, which claims
the benefit of U.S. Ser. No. 61/280,510, filed Nov. 4, 2009;
(2) U.S. patent application Ser. No. 14/353,460, which is a
continuation of PCT/CA2012/000991, filed Oct. 25, 2012, which
claims the benefit of U.S. Ser. No. 61/551,366, filed Oct. 25,
2011;
(3) PCT/US2014/029116, filed Mar. 14, 2014 (published as
WO2014172045, Oct. 23, 2014), which claims the benefit of U.S. Ser.
No. 61/798,495, filed Mar. 15, 2013;
(4) PCT/US2014/041865, filed Jul. 25, 2014 (published as
WO2015013596, Jan. 29, 2015), which claims the benefit of U.S. Ser.
No. 61/858,973, filed Jul. 26, 2013; and
(5) PCT/US2014/060961, which claims the benefit of U.S. Ser. No.
61/891,758, filed Oct. 16, 2013; and
(6) U.S. Provisional Patent Application No. 62/120,179, filed Feb.
24, 2015, the disclosures of which are hereby incorporated by
reference in their entirety.
Microfluidic Cartridge
In one aspect, a microfluidic cartridge is provided that is
disposable and is configured to mix a first solution with a second
solution to produce a mixed solution. In one embodiment, the
microfluidic cartridge includes:
(A) a carrier, comprising a first inlet connector, a second inlet
connector, and an outlet opening;
(B) a first microfluidic structure integrally incorporated into the
carrier, the microfluidic structure comprising:
(I) a first inlet microchannel configured to receive fluid from
first inlet connector;
(II) a second inlet microchannel configured to receive fluid from
the second inlet connector;
(III) a first mixer configured to:
receive a first solution from the first inlet microchannel and a
second solution from the second inlet microchannel;
mix the first solution and the second solution to provide a mixed
solution, and
direct the mixed solution into an outlet microchannel; and
(IV) the outlet microchannel, which is in fluid communication with
the outlet opening.
Embodiments of the microfluidic cartridge will now be described
with reference to FIGURES, where like numbers indicate like parts.
Referring to FIG. 1A, a microfluidic cartridge 100 includes a
carrier 105 that includes a first inlet connector 110, a second
inlet connector 115, and an outlet opening 120. The inlet
connectors 110 and 115 are configured to form a fluid-tight
connection with sources of solution to be mixed in the cartridge
100.
Sources of solution (also referred to as "reservoirs") include
syringes and pumps. The microfluidic cartridge can be made
compatible with any source of solution by way of appropriate
configuration of the inlet connectors 110 and 115 to mate with the
opposite connectors attached to the sources of solution.
The illustrated inlet connectors 110 and 115 are in the form of
tapered connectors, but it will be appreciated that any fluidic
connector type can be used, including Luer-Lok connectors, as
illustrated in FIG. 1C. In one embodiment, the inlet connectors 110
and 115 are Luer fittings designed to be compatible with ISO 594.
The dimensions of the inlet connectors 110 and 115 may be optimized
to reduce the internal volume. In one embodiment, the inlet
connectors 110 and 115 provide a sterile or aseptic fluid path for
the inlet fluid from the fluid vessel to the microfluidic structure
125.
The outlet opening 120 is configured to allow fluid to flow from
the microfluidic cartridge 100 into an outlet receptacle. In one
embodiment of the microfluidic cartridge, the outlet opening 120 is
a nozzle that directs the fluid to a receptacle below. In one
embodiment, the outlet opening 120 fitting provides a hermetic seal
to the outlet receptacle. An example of such a fitting would be a
Luer fitting compatible with ISO 594. In one embodiment, the outlet
opening 120 provides a sterile or aseptic fluid path for the outlet
stream from the microfluidic cartridge to the outlet
receptacle.
While tapered connectors are illustrated in the FIGURES, it will be
appreciated that any connection type can be used as long as fluid
can be passed through the connector and into (or away from) the
microfluidic structure 125. Accordingly, in another embodiment, the
inlet connectors are gaskets (e.g., O-rings) configured to form a
pressure seal with a connector to a source of fluid (e.g., a pump
head) off of the cartridge 100. In a further embodiment, the
gaskets are fitted in seating grooves formed in the surface of the
microfluidic cartridge 100. In certain embodiment, the outlet
connector is a gasket, similar to the previously described inlet
connectors.
Referring to FIG. 1B, a microfluidic structure 125 is disposed
within the carrier 105. In the illustrated embodiment, the
microfluidic structure 125 is in the form of a chip that is
distinct from the carrier 105. The microfluidic structure 125
includes a first inlet microchannel 130 configured to receive fluid
(e.g., a first solution) from first inlet connector 110; and a
second inlet microchannel 135 configured to receive fluid (e.g., a
second solution) from the second inlet connector 115. A first mixer
140 is configured to receive flow from the first inlet microchannel
130 from a first transport microchannel 131 and from the second
inlet microchannel 135 from a second transport microchannel 136.
The first mixer is configured to mix two fluids to form a mixed
solution and deliver the mixed solution to the outlet opening 120
via a third transport microchannel 146 and an outlet microchannel
145.
The first mixer 140 is a "microfluidic element," which is defined
herein as a microfluidic component configured to perform a function
beyond simply flowing solution, such as mixing, heating, filtering,
reacting, etc. In several exemplary embodiments disclosed herein,
the microfluidic elements described are microfluidic mixers
configured to mix a first solution with a second solution in a
mixer to provide a mixed solution. However, other microfluidic
devices are also compatible with the disclosed embodiments.
While only a single microfluidic element (mixer 140) is
illustrated, it will be appreciated that multiple microfluidic
elements can be incorporated into the microfluidic cartridge 100.
In certain embodiments, this includes a second mixer. Further inlet
connections may also be added in order to support functions of the
additional microfluidic elements. In one embodiment, a plurality of
mixers (microfluidic elements) are included in the microfluidic
structure.
For example, in another embodiment (not illustrated) a third inlet
connection is included and a second mixer is included to perform a
dilution of the mixed solution produced by the first mixer 140 by
mixing a dilution solution provided via the third inlet
connector.
The microfluidic structure 125 is integrally incorporated into the
carrier 105. As used herein, the term "integrally incorporated"
refers to a microfluidic structure that is not readily removable
from the carrier. For example, a microfluidic structure is
integrally incorporated into a carrier if the carrier is only
openable--to expose the microfluidic structure--with a tool (e.g.,
a screwdriver used to unfasten securing screws). Additionally, a
microfluidic structure is integrally incorporated into a carrier if
the carrier is sealed shut, such that the microfluidic structure
could only be removed by breaking the carrier. As a final example,
a microfluidic structure is integrally incorporated into a carrier
if the microfluidic structure is physically attached or part of the
carrier (e.g., if the microfluidic cartridge is of monolithic
construction or has been permanently adhered using an adhesive,
solvent weld or other technique). Such a monolithic construction is
not considered to incorporate a microfluidic chip, because the
microfluidic structure is a part of an element of the carrier that
provides a function besides microfluidic flow (e.g., structural
support).
In a further embodiment, being integrally incorporated indicates
that the microfluidic cartridge cannot be taken apart and put back
together again. For example, the microfluidic structure cannot be
removed from the carrier and then replaced and sealed.
The microfluidic cartridge 100 is disposable. As used herein the
term "disposable" refers to a component that has relatively low
cost in relation to the product produced by the microfluidic
cartridge (e.g., nano-medicine). Furthermore, a disposable
microfluidic cartridge has a limited useful life, such as only
being fit for single use, as described below. Disposable materials
broadly include plastics, magnets (e.g., inorganic materials), and
metals.
In one embodiment, the microfluidic cartridge is configured for a
single use. In this regard, the microfluidic cartridge is of a
construction that results in low manufacturing cost and therefore
allows a user to dispose of the cartridge after use. In certain
embodiments, a property of the cartridge is altered after a single
use, therefore discouraging or eliminating the possibility of
further uses of the cartridge. For example, with regard to a
sterile cartridge (as described below), after a single use the
cartridge is no longer sterile and therefore would not be usable
again as a sterile cartridge. Furthermore, a single use cartridge
eliminates the risk of cross-contamination between mixings. In this
regard, a single-use microfluidic cartridge contains an entirely
unused (untouched by fluid) fluidic path, from inlet connectors to
outlet. Existing chip/holder technologies risk cross contamination
due to the inlet connectors and outlet being reused between
mixings. As used herein, the term "chip" refers to a freestanding
microfluidic layer that is subsequently integrated into a holder
containing inlet/outlet connections. The disclosed microfluidic
cartridges are distinct from such chip/holder systems by integrally
incorporating a microfluidic structure--which is a chip in certain
embodiments, but is not a chip in other embodiments.
In one embodiment, the carrier comprises a first portion,
comprising the first inlet connector, the second inlet connector,
and the outlet opening and a second portion, wherein the first
portion and the second portion join to seal the microfluidic
structure between the first portion and the second portion. In
certain embodiments herein, the first portion of the carrier may be
referred to as the connection portion and the second portion may be
referred to as the top plate. Certain embodiments may require the
use of additional components, such as screws and plates, to
complete the coupling between the first and second portions of the
carrier. In one embodiment, referring to FIG. 2, the second portion
150 serves to apply a clamping force to the assembly. In one
embodiment, the second portion 150 contains a layer or mechanism to
evenly distribute clamping forces across the microfluidic
structure.
Referring now to FIGS. 2 and 3, a representative embodiment of a
microfluidic cartridge is illustrated in exploded view in order to
more easily view how the microfluidic cartridge is assembled. In
the illustrated embodiment, the carrier 105 is split into two
portions, with the carrier 105 being a first portion that joins
with a second portion 150 to seal the microfluidic structure 125
therebetween. The two carrier portions 105 and 150 are integrally
joined by a plurality of fasteners 155 (illustrated as screws).
Accordingly, in one embodiment, the first portion and the second
portion of the carrier are secured together by one or more
fasteners. In one embodiment, the one or more fasteners are
removable. Exemplary removable fasteners are screws, nuts and
bolts, clips, straps, and pins.
In another embodiment, the one or more fasteners are not removable.
In such embodiments, the fasteners may be nails or rivets. In an
additional embodiment, such fasteners may be incorporated as a
feature of the carrier. In such and embodiment, one portion may
contain clips or tabs with a second portion having recesses,
notches or other mechanisms to receive such a fastener.
In another embodiment, the first portion and the second portion are
bonded together. In such embodiments, the two portions are not
separable once joined. In one embodiment, the first portion and the
second portion are bonded together with an adhesive. In one
embodiment, the first portion and the second portion are bonded
together with a weld. Representative compatible welding methods
include laser welding, ultrasonic welding, and solvent welding.
Referring again to FIGS. 2 and 3, the microfluidic cartridge
further includes gaskets 160 configured to form separate
fluid-tight seals between the microfluidic structure 125 and the
first inlet connector 110, the second inlet connector 115, and the
outlet opening 125. As illustrated in FIG. 3, the gaskets 160 are
nested in recesses 161 in the first portion of the carrier 105.
While gaskets in the form of O-rings are illustrated, in certain
embodiments, flanges or other feature integrated into the carrier
105 are utilized to form the required seal.
Securing Mechanism
In one embodiment, the microfluidic cartridge further includes a
securing mechanism configured to secure the microfluidic cartridge
to a holder. In one embodiment the holder is an apparatus
configured to arrange the microfluidic cartridge in relation to
fluid sources (e.g., syringes) and to facilitate connections
between them. An exemplary holder is illustrated or pictured in
FIGS. 5A, 5C, and 5H, as will be discussed in further detail
below.
In the embodiment illustrated in FIGS. 2 and 3, the securing
mechanism comprises multiple magnets 165 placed in recesses within
the carrier 105. The magnets 165 attract to metal or opposite
magnets on the holder in order to secure the microfluidic cartridge
100 for use.
In another embodiment, the securing mechanism comprises a carrier
locking feature on the carrier that is configured to lock with a
holder locking feature on a compatible holder for the microfluidic
cartridge. Such a holder locking feature may include a recess in
the carrier that mates with or is otherwise secured by an arm or
other projection attached to the holder. An example of such a
locking feature would be a leaf spring with a matching recess.
Cartridge Materials and Construction
The carrier and microfluidic structure are formed from materials
capable of being formed into the necessary shapes and with the
necessary physical characteristics. The materials are disposable
and therefore relatively inexpensive. The material of the
microfluidic structure is capable of being formed into the
necessary micron-sized microfluidic elements and then withstanding
the pressures applied during mixing within the microfluidic
structure. The material of the carrier is sufficiently rigid that
it will protect and support the microfluidic structure within the
carrier.
In one embodiment, the microfluidic structure is formed from a
material different than that of the carrier. In another embodiment,
the microfluidic structure and the carrier are formed from the same
material. In a further embodiment, the microfluidic structure and
the carrier are monolithically formed.
In one embodiment, the carrier contains no metal. In another
embodiment, the carrier may contain some metal, but the carrier is
at least 90%, by weight, polymer. In one embodiment, the carrier
contains no metal. In another embodiment, the carrier may contain
some metal, but the carrier is at least 99%, by weight,
polymer.
In one embodiment, the carrier comprises a polymer selected from
the group consisting of polypropylene, polycarbonate, COC, COP,
polystyrene, nylon, acrylic, HPDE, LPDE, and other polyolefins.
In one embodiment, the carrier does not include metal on an
exterior surface. Such an embodiment contemplates the potential
presence of magnets or other metal-containing elements within the
carrier, but not on the exterior surface.
In another embodiment, the first inlet connector and the second
inlet connector are formed from a polymer. In certain embodiments
it is preferable that the inlet connectors be formed from
relatively soft polymer material, particularly when a taper or Luer
connector is used. A softer polymer will ameliorate minor
manufacturing errors of the inlets and allow a fluid-tight
connection to be made. More rigid polymers will not allow for such
a forgiving characteristic. In this regard, in one embodiment, the
first inlet connector comprises a polymer having a Young's modulus
of 500 MPa to 3500 MPa. In one embodiment, the first inlet
connector comprises a polymer having a Young's modulus of 2000 MPa
to 3000 MPa.
In one embodiment, the second inlet connector comprises a polymer
having a Young's modulus of 500 MPa to 3500 MPa. In one embodiment,
the second inlet connector comprises a polymer having a Young's
modulus of 2000 MPa to 3000 MPa.
In one embodiment, the carrier is formed from a polymer having a
Young's modulus of 500 MPa to 3500 MPa. In one embodiment, the
carrier comprises a polymer having a Young's modulus of 2000 MPa to
3000 MPa.
In one embodiment, the carrier comprises a metal selected from the
group consisting of aluminum and steel. As noted above, in certain
embodiments, small amounts of metal can be incorporated into the
carrier. In such a situation, the microfluidic cartridge remains
disposable.
In one embodiment, the microfluidic structure is not separable from
the carrier. In such an embodiment, the microfluidic structure is
attached (e.g., welded or adhered to) at least a portion of the
carrier. In one embodiment the microfluidic cartridge is monolithic
in construction, with the carrier and microfluidic structure being
formed from the same material. In a further embodiment, the
microfluidic cartridge is composed of at least two parts (e.g., a
connection portion and a top plate) with the microfluidic structure
being incorporated into one of the two parts. That is, the
microfluidic structure is attached (e.g., bonded or welded to) to a
portion of the microfluidic cartridge that performs an addition
function beyond providing microfluidic elements. In one embodiment
the microfluidic structure is attached to the top plate. In a
further embodiment, the microfluidic structure and the top plate
are monolithic and formed from the same material. In yet a further
embodiment, the microfluidic structure is of monolithic
construction with one of the two parts.
In one embodiment, the carrier encloses the microfluidic structure.
As used herein, the term "encloses" indicates that the carrier
surrounds a majority of the surface area of the microfluidic
structure. Of primary importance is that the carrier facilitates a
fluid-tight seal with the microfluidic structure and provide a
rigid housing that allows for handling of the microfluidic
cartridge. In a further embodiment, the carrier entirely encloses
the microfluidic structure, meaning that no surface area of the
microfluidic structure is exposed outside the carrier. Such an
embodiment is illustrated in FIGS. 1A-3.
In one embodiment, the first portion and the second portion join to
enclose the microfluidic structure. Such an embodiment is
illustrated in FIGS. 2 and 3.
In one embodiment, the first portion is at least 90%, by weight,
polymer. In this embodiment, the first portion includes the inlet
connectors and outlet opening.
In one embodiment, the first portion or the second portion
comprises the microfluidic structure. In such an embodiment, the
microfluidic structure is attached to, or monolithic with, the
first portion or the second portion of the carrier.
Fluid Sources
The fluid or solution reservoirs are selected such as to allow a
direct connection to the microfluidic cartridge. In one embodiment,
the fluid reservoirs are disposable syringes. In a further
embodiment, the fluid reservoirs are prefilled syringes. Both the
fluid and the reservoir may be sterile in order to produce sterile
nanoparticles. The system contains a means by which to cause the
fluid to flow from the reservoir and through the cartridge at a
prescribed flow rate. In one embodiment of the system, the fluid is
caused to flow by pressurizing the reservoirs causing a first and
second stream to enter the cartridge (through the inlets into the
microfluidic structure and its channels). Examples of means of
pressurization include, but are not limited to, linear actuators
and inert gas. In one embodiment, each reservoir is pressurized
independently. In one embodiment, two or more reservoirs are
pressurized by the same source and differential flow rates are
achieved by varying the dimensions of the fluidic channels.
Differing flow ratios may be enabled by either differential
pressure drops across the flow channels, differential channel
impedances, or combination therein, applied to the inlet streams.
Differential impedances of the channels through varying the channel
heights, widths, lengths, or surface properties, may be used to
achieve different flow rates. Fluidic surface tensions,
viscosities, and other surface properties of the flows in the one
or more first streams and the one or more second streams may be
used or considered to achieve different flow rates. Pressurization
of the vessels may be controlled using a computer or
microcontroller.
In certain embodiments, the system further includes means for
complete or partial purging of the system to minimize the waste
volume. After or during manufacture of particles, purging may be
achieved by flowing a gas or liquid through the fittings and
microfluidic structure. Gasses such as air, nitrogen, argon or
others may be used. Liquids including water, aqueous buffer,
ethanol, oils, or any other liquid may be used.
Microfluidic Mixers
In one embodiment, the first mixer comprises a mixing region
comprising a microfluidic mixer configured to mix the first
solution and the second solution to provide a mixed solution. Such
microfluidic mixers are generally known to those of skill in the
art and exemplary mixers are disclosed in the patent documents
incorporated herein by reference.
In one embodiment, the first mixer is a chaotic advection
mixer.
In one embodiment, the mixing region comprises a herringbone
mixer.
In one embodiment, the mixing region has a hydrodynamic diameter of
about 20 microns to about 300 microns.
In one embodiment, the first mixer is sized and configured to mix
the first solution and the second solution at a Reynolds number of
less than 1000.
In one embodiment, the microfluidic structure further comprises a
plurality of mixers in series.
In certain aspects, the disposable cartridge contains a
microfluidic component which is a microfluidic mixer to rapidly and
controllably mix two or more streams. Many methods for this mixing
process are known. In one embodiment, the mixing is chaotic
advection. Compatible microfluidic mixing methods and devices are
disclosed in the patent applications incorporated herein by
reference. Furthermore, representative microfluidic devices are
disclosed in further detail herein. In certain embodiments, devices
are provided for making nanoparticles of the type disclosed herein.
The microfluidic devices are incorporated into the disposable
cartridge and methods disclosed herein.
In one embodiment, with reference to FIG. 4, the microfluidic
structure includes:
(a) a first inlet 302 for receiving a first solution comprising a
first solvent;
(b) a first inlet microchannel 304 in fluid communication with the
first inlet to provide a first stream comprising the first
solvent;
(c) a second inlet 306 for receiving a second solution comprising
lipid particle-forming materials in a second solvent;
(d) a second inlet microchannel 308 in fluid communication with the
second inlet to provide a second stream comprising the lipid
particle-forming materials in the second solvent; and
(e) a third microchannel 310 for receiving the first and second
streams, wherein the third microchannel has a first region 312
adapted for flowing the first and second streams and a second
region 314 adapted for mixing the contents of the first and second
streams to provide a third stream comprising a mixed solution. In
one application of the microfluidic cartridge, the mixed solution
comprises limit size lipid nanoparticles. The lipid nanoparticles
so formed are conducted from the second (mixing) region by
microchannel 316 to outlet 318.
In one embodiment, the second region of the microchannel comprises
bas-relief structures. In certain embodiments, the second region of
the microchannel has a principal flow direction and one or more
surfaces having at least one groove or protrusion defined therein,
the groove or protrusion having an orientation that forms an angle
with the principal direction. In other embodiments, the second
region includes a micromixer.
In the devices and systems, means for varying the flow rates of the
first and second streams are used to rapidly mix the streams
thereby providing the nanoparticles.
In certain embodiments, the devices of the disclosure provide
complete mixing occurs in less than 10 ms.
In certain embodiments, one or more regions of the device are
heated.
In one embodiment, the first mixer comprises a mixing region
comprising a microfluidic mixer configured to mix the first
solution and the second solution to provide the nanoparticle
solution formed from mixing of the first solution and the second
solution.
In one embodiment, the first mixer is a chaotic advection
mixer.
In one embodiment, the mixing region comprises a herringbone
mixer.
While a staggered herringbone mixer (SHM) is illustrated in certain
FIGURES (e.g., FIG. 4A), it will be appreciated that other mixing
configurations are also contemplated. In one embodiment, the mixer
is a dean vortexing mixer. In another embodiment, the mixer is a
Dean vortex bifurcating mixer (DVBM), which are discussed in
greater detail below. In one embodiment, the microfluidic structure
includes two different types of chaotic advection mixers. In a
further embodiment, the two different types of chaotic advection
mixers are SHM and Dean vortexing. In one embodiment, the
microfluidic structure includes two different types of chaotic
advection mixers, wherein at least one of the two chaotic advection
mixers is selected from the group consisting of SHM and Dean
vortexing.
In one embodiment, the mixing region has a hydrodynamic diameter of
about 20 microns to about 300 microns. In one embodiment, the
mixing region has a hydrodynamic diameter of about 113 microns to
about 181 microns. In one embodiment, the mixing region has a
hydrodynamic diameter of about 150 microns to about 300 microns. As
used herein, hydrodynamic diameter is defined using channel width
and height dimensions as (2*Width*Height)/(Width+Height).
The mixing region can also be defined using standard width and
height measurements. In one embodiment, the mixing region has a
width of about 100 to about 500 microns and a height of about 50 to
about 200 microns. In one embodiment, the mixing region has a width
of about 200 to about 400 microns and a height of about 100 to
about 150 microns.
In order to maintain laminar flow and keep the behavior of
solutions in the microfluidic devices predictable and the methods
repeatable, the systems are designed to support flow at low
Reynolds numbers. In one embodiment, the first mixer is sized and
configured to mix the first solution and the second solution at a
Reynolds number of less than 2000. In one embodiment, the first
mixer is sized and configured to mix the first solution and the
second solution at a Reynolds number of less than 1000. In one
embodiment, the first mixer is sized and configured to mix the
first solution and the second solution at a Reynolds number of less
than 900. In one embodiment, the first mixer is sized and
configured to mix the first solution and the second solution at a
Reynolds number of less than 500.
In one embodiment, the microfluidic mixer device contains one
micromixer. In one embodiment, the single mixer microfluidic device
has two regions: a first region for receiving and flowing at least
two streams (e.g., one or more first streams and one or more second
streams). The contents of the first and second streams are mixed in
the microchannels of the second region, wherein the microchannels
of the first and second regions has a hydrodynamic diameter from
about 20 to about 500 microns. In a further embodiment, the second
region of the microchannel has a principal flow direction and one
or more surfaces having at least one groove or protrusion defined
therein, the groove or protrusion having an orientation that forms
an angle with the principal direction (e.g., a staggered
herringbone mixer), as described in US 2004/0262223, expressly
incorporated herein by reference in its entirety. In one
embodiment, the second region of the microchannel comprises
bas-relief structures. In certain embodiments, the second regions
each have a fluid flow rate of from 1 to about 50 mL/min. In a
preferred embodiment, the mixing channel of the microfluidic device
is 300 microns wide and 130 microns high. In other embodiments, the
first and second streams are mixed with other micromixers. Suitable
micromixers include droplet mixers, T-mixers, zigzag mixers,
mulitlaminate mixers, or other active mixers.
One function of the systems and methods disclosed herein is to form
nanoparticles in solution (the "product"). Previous disclosures by
the present inventors relate to generating nanoparticles compatible
with the present system, such as the patent applications
incorporated herein by reference. Known and future-developed
nanoparticle methods can be performed on the disclosed systems to
the extent that the methods require the controlled combination of a
first solution with a second solution to form a nanoparticle
product, as disclosed herein.
The first solution, also referred to herein as the "aqueous
reagent" herein, is provided in a first solution reservoir. In one
embodiment, the first solution comprises a first solvent. In one
embodiment, the first solution comprises an active pharmaceutical
ingredient. In one embodiment, the first solution comprises a
nucleic acid in a first solvent. In another embodiment, the first
solution comprises a buffer. In one embodiment, the first solution
consists essentially of a buffer.
The second solution, also referred to herein as the "solvent
reagent" herein, is provided in a second solution reservoir. In one
embodiment, the second solution comprises a second solvent. In one
embodiment, the second solution comprises lipid particle-forming
materials in a second solvent. In one embodiment, the second
solvent is a water-miscible solvent (e.g., ethanol or
acetonitrile). In certain embodiments, the second solution is an
aqueous buffer comprising polymer nanoparticle forming
reagents.
In one embodiment, the first solution comprises a nucleic acid in a
first solvent and the second solution comprises lipid
particle-forming materials in a second solvent.
Dean Vortex Bifurcating Mixers ("DVBM")
As noted above, DVBM are useful as mixers in the disclosed
microfluidic cartridges. DVBMs of the type disclosed herein act as
efficient mixers and whose injection molding tooling can be
produced by an end mill with a radius of R (for example 300 .mu.m).
The provided DVBM mixers include a plurality of toroidal mixing
elements (also referred to herein as "toroidal mixers"). As used
herein, "toroid" refers to a generally circular structure having
two "leg" channels that define a circumference of the toroid
between an inlet and an outlet of the toroidal mixer. The toroidal
mixers are circular in certain embodiments. In other embodiments,
the toroidal mixers are not perfectly circular and may instead have
oval or non-regular shape.
FIG. 4B illustrates a pair of toroidal DVBM mixers in accordance
with the disclosed embodiments. FIG. 4C is a photograph of an
exemplary toroidal DVBM mixer in accordance with the disclosed
embodiments.
In one embodiment, the DVBM mixer is configured to mix at least a
first liquid and a second liquid, the mixer comprising an inlet
channel leading into a plurality of toroidal mixing elements
arranged in series, wherein the plurality of toroidal mixing
elements includes a first toroidal mixing element downstream of the
inlet channel, and a second toroidal mixing element in fluidic
communication with the first toroidal mixing element via a first
neck region, and wherein the first toroidal mixing element defines
a first neck angle between the inlet channel and the first neck
region.
In one embodiment, the first neck angle is from 0 to 180
degrees.
In one embodiment, the first neck region has a length of 0.2 mm or
greater.
In one embodiment, the plurality of mixing elements include
channels having a hydrodynamic diameter of about 20 microns to
about 2 mm.
In one embodiment, the mixer is sized and configured to mix the
first liquid and the second liquid at a Reynolds number of less
than 1000.
In one embodiment, the mixer includes two or more mixers in
parallel, each mixer having a plurality of toroidal mixing
elements.
In one embodiment, the first toroidal mixing element and the second
toroidal mixing element define a mixing pair, and wherein the mixer
includes a plurality of mixing pairs, and wherein each mixing pair
is joined by a neck region at a neck angle.
In one embodiment, the first toroidal mixing element has a first
leg of a first length and a second leg of a second length; and
wherein the second toroidal mixing element has a first leg of a
third length and a second leg of a fourth length.
In one embodiment, the first length is greater than the second
length.
In one embodiment, the third length is greater than the fourth
length.
In one embodiment, the ratio of the first length to the second
length is about equal to the ratio of the third length to the
fourth length.
In one embodiment, the first toroidal mixing element has a first
leg of a first impedance and a second leg of a second impedance;
and wherein the second toroidal mixing element has a first leg of a
third impedance and a second leg of a fourth impedance.
In one embodiment, the first impedance is greater than the second
impedance.
In one embodiment, the third impedance is greater than the fourth
impedance.
In one embodiment, the ratio of the first impedance to the second
impedance is about equal to the ratio of the third impedance to the
fourth impedance.
In one embodiment, the mixer includes 2 to 20 toroidal mixing
elements in series.
In one embodiment, the mixer includes 1 to 10 pairs of toroidal
mixing elements in series.
In one embodiment, the toroidal mixing elements have an inner
radius of about 0.1 mm to about 2 mm.
Also provided are methods of mixing a first liquid with a second
liquid using a microfluidic cartridge as disclosed herein, the
method comprising flowing the first liquid and the second liquid
through a DVBM mixer according to the disclosed embodiments.
Sterility
A sterile cartridge is essential for certain applications and
provides a convenient workflow for users to directly formulate
sterile nanoparticles without the need for further filtration or
treatment. Such a workflow minimizes the material loss associated
with further sterilization steps. In one embodiment, the individual
components of the cartridge are sterilized prior to assembly.
Representative sterilization methods include steam autoclave, dry
heat, chemical sterilization (i.e. sodium hydroxide or ethylene
oxide), gamma radiation, gas and combinations thereof. In a
specific embodiment, the microfluidic structure, inlet fittings,
outlet fitting and any other fluid contact components are formed
from materials that are compatible with gamma radiation and are
sterilized by such means. Materials compatible with gamma radiation
are those that can be irradiated. For example, polycarbonate,
cyclic olefin polymer, cyclic olefin copolymer, polypropylene, and
high- and low-density polyethylene. Materials that cannot be
irradiated include polyamides, polytetrafluoroethylene, and any
metal. In a further embodiment, the cartridge is sterilized after
assembly.
In one embodiment, the cartridge is sterilizable. As used herein,
the term "sterilizable" refers to a cartridge formed from materials
that are compatible with known sterilization methods, as set forth
above. In one embodiment, the cartridge is specifically
sterilizable by gamma radiation. In a further embodiment, the
cartridge is formed from a polymer selected from the group
consisting of polypropylene, polycarbonate, a cyclic olefin
polymer, a cyclic olefin copolymer, high-density polyethylene,
low-density polyethylene, and combinations thereof. In a further
embodiment, the cartridge does not include polyamides,
polytetrafluoroethylene, or any metal.
In one embodiment, the microfluidic cartridge is sterile.
In one embodiment, the microfluidic cartridge includes a sterile
fluid path, from the first inlet connector and the second inlet
connector, through the microfluidic structure, and to the outlet
opening. Such a sterile fluid path allows for mixing in a sterile
environment. Because the inlet connectors and outlet opening are
also sterile, sterile connections can be easily facilitated.
In another aspect, a sterile package filled with sterile contents
is provided. In one embodiment, the sterile package includes a
microfluidic cartridge according to any of the embodiments
disclosed herein in a sterile state and sealed within the sterile
package. A sterile package is defined by an enclosure containing
sterile contents. The enclosure is a bag in one embodiment. By
providing the microfluidic cartridge in a sterile state and sealed
within the sterile package, an end user can easily perform sterile
microfluidic mixing using the cartridge by opening the sterile
package in a sterile environment and using it for mixing without
any further preparation. No sterilization is needed for any of the
inlet connectors or the fluid path, which are sterile.
In one embodiment, the sterile package further includes a first
sterile syringe configured to couple with the first inlet connector
of the microfluidic cartridge. In such an embodiment, the sterile
package is a kit that includes both the microfluidic cartridge and
a sterile syringe configured for use with the microfluidic
cartridge. In one embodiment, the sterile package further includes
a first solution within the first sterile syringe.
In one embodiment, the first solution comprises a nucleic acid in a
first solvent. In a further embodiment, the first solution is of
the type configured for use to form lipid nanoparticles.
In one embodiment, the sterile package further includes a second
sterile syringe configured to couple with the second inlet
connector of the microfluidic cartridge.
In one embodiment, the sterile package further includes a second
solution within the second sterile syringe.
In one embodiment, the second solution comprises lipid
particle-forming materials in a second solvent. Such a second
solution can be combined with a first solution comprising a nucleic
acid in a first solvent in order to form a lipid nanoparticle
solution via the microfluidic cartridge.
In one embodiment, the sterile package further includes a sterile
receptacle configured to couple with the outlet opening of the
microfluidic cartridge via an outlet opening connector.
In one embodiment, the sterile contents are disposable.
Methods of Using the Microfluidic Cartridge
In another aspect, a method of forming nanoparticles is provided.
In one embodiment, the method includes flowing a first solution and
a second solution through a microfluidic cartridge according to any
of the disclosed embodiments and forming a nanoparticle solution in
the first mixer.
Methods of forming nanoparticles using microfluidic mixers is
generally known in the art and these methods are applicable to the
disclosed microfluidic cartridges, which essentially provide an
improved and simplified manner for performing known methods.
Exemplary methods are disclosed in the patent documents
incorporated herein by reference. The Example below describes a
specific method for generating siRNA lipid nanoparticles using an
exemplary microfluidic cartridge.
In one embodiment, the first solution comprises a nucleic acid in a
first solvent.
In one embodiment, the second solution comprises lipid
particle-forming materials in a second solvent.
In one embodiment, the microfluidic cartridge comprises a plurality
of mixers and the method further comprises flowing the first
solution and the second solution through the plurality of mixers to
form the nanoparticle solution, wherein the plurality of mixers
includes the first mixer. Such embodiments contemplate the
introduction of a third solution for dilution of the mixed solution
(e.g., to stabilize a lipid nanoparticle solution formed in the
first mixer). Another representative use of a third, or subsequent,
mixer, is the addition of further components to the mixed solution,
such as a targeting ligand for a lipid nanoparticle that has
already been created in the first mixer.
The disclosed methods can be facilitated by an apparatus for
holding and/or manipulating the microfluidic cartridge. In this
regard, FIG. 5A illustrates an embodiment of microfluidic system
500 that includes a cartridge holder 505 configured to facilitate
connections between a microfluidic cartridge 100, specifically
inlet connectors 110 and 115, and syringes 510 and 515,
respectively, in order to mix solutions contained therein and
deliver the mixed solution to the outlet opening 120 of the
microfluidic cartridge 100. In the illustrated embodiment, a clamp
520 is provided to support a collection vial (not illustrated) to
collect mixed solution produced at the outlet opening 120.
The holder 505 includes a mechanism (not illustrated) for securing
the cartridge 100. Such securing mechanisms are disclosed elsewhere
herein and include magnets within the cartridge 100 (see part 165
in FIGS. 2 and 3) configured to produce magnetic attraction to a
portion of the holder 505 sufficient to immobilize the cartridge
100 in position.
FIGS. 5C and 5H are pictures depicting a microfluidic system
including a holder facilitating a connection between a microfluidic
cartridge and two syringes. The pictured holder also includes
platforms configured to operate the syringes and facilitate
mixing.
Methods of using the microfluidic cartridge also include methods
performed in a sterile environment, as is desirable when forming
certain nanoparticles (e.g., nano-medicines). Accordingly, in one
embodiment, the microfluidic cartridge has a sterile fluid path.
Such embodiments and advantages are described above with regard to
the microfluidic cartridge.
In one embodiment, the method further includes a step of
sterilizing the fluid path prior to the step of flowing the first
solution and the second solution through the microfluidic
cartridge.
In one embodiment, the step of sterilizing the fluid path comprises
sterilizing the microfluidic cartridge with radiation.
In one embodiment, the step of sterilizing the fluid path comprises
sterilizing portions of the microfluidic cartridge prior to
assembling the microfluidic cartridge.
In one embodiment, the sterile fluid path comprises a fluidic path
from the first inlet connector and the second inlet connector,
through the microfluidic structure, and to the outlet opening.
In one embodiment, the sterile fluid path further comprises a first
syringe, containing the first solution, coupled to the first
inlet.
In one embodiment, the sterile fluid path further comprises a
second syringe, containing the second solution, coupled to the
second inlet.
In one embodiment, the sterile fluid path further comprises a
sterile receptacle coupled with the outlet opening of the
microfluidic cartridge via an outlet opening connector, and wherein
the method further comprises a step of delivering the nanoparticle
solution from the mixer to the sterile receptacle via the outlet
microchannel and outlet opening.
In one embodiment, the method does not include a step of integrally
incorporating the microfluidic structure into the carrier. One of
the advantages of the microfluidic cartridge is the lack of need to
assemble a microfluidic chip into a carrier. Therefore, when
performing the method according to this embodiment, no assembly of
the microfluidic cartridge is performed.
In one embodiment, the method further includes a step of removing
the microfluidic cartridge from a sterile package prior to the step
of flowing the first solution and the second solution through the
microfluidic cartridge. With regard to sterile methods of using the
microfluidic cartridges, and when the microfluidic cartridge is
provided in a sterile package, the method includes a step of
removing the microfluidic cartridge from the sterile package prior
to its use to mix a solution.
Definitions
Microfluidic
As used herein, the term "microfluidic" refers to a system or
device for manipulating (e.g., flowing, mixing, etc.) a fluid
sample including at least one channel having micron-scale
dimensions (i.e., a dimension less than 1 mm).
Therapeutic Material
As used herein, the term "therapeutic material" is defined as a
substance intended to furnish pharmacological activity or to
otherwise have direct effect in the diagnosis, cure, mitigation,
understanding, treatment or prevention of disease, or to have
direct effect in restoring, correcting or modifying physiological
functions. Therapeutic material includes but is not limited to
small molecule drugs, nucleic acids, proteins, peptides,
polysaccharides, inorganic ions and radionuclides.
Nanoparticles
As used herein, the term "nanoparticles" is defined as a
homogeneous particle comprising more than one component material
(for instance lipid, polymer etc.) that is used to encapsulate a
therapeutic material and possesses a smallest dimension that is
less than 250 nanometers. Nanoparticles include, but are not
limited to, lipid nanoparticles and polymer nanoparticles.
Lipid Nanoparticles
In one embodiment, lipid nanoparticles, comprise:
(a) a core; and
(b) a shell surrounding the core, wherein the shell comprises a
phospholipid.
In one embodiment, the core comprises a lipid (e.g., a fatty acid
triglyceride) and is solid. In another embodiment, the core is
liquid (e.g., aqueous) and the particle is a vesicle, such as a
liposomes. In one embodiment, the shell surrounding the core is a
monolayer.
As noted above, in one embodiment, the lipid core comprises a fatty
acid triglyceride. Suitable fatty acid triglycerides include C8-C20
fatty acid triglycerides. In one embodiment, the fatty acid
triglyceride is an oleic acid triglyceride.
The lipid nanoparticle includes a shell comprising a phospholipid
that surrounds the core. Suitable phospholipids include
diacylphosphatidylcholines, diacylphosphatidylethanolamines,
ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and
cerebrosides. In one embodiment, the phospholipid is a C8-C20 fatty
acid diacylphosphatidylcholine. A representative phospholipid is
1-palmitoyl-2-oleoyl phosphatidylcholine (POPC).
In certain embodiments, the ratio of phospholipid to fatty acid
triglyceride is from 20:80 (mol:mol) to 60:40 (mol:mol).
Preferably, the triglyceride is present in a ratio greater than 40%
and less than 80%.
In certain embodiments, the nanoparticle further comprises a
sterol. Representative sterols include cholesterol. In one
embodiment, the ratio of phospholipid to cholesterol is 55:45
(mol:mol). In representative embodiments, the nanoparticle includes
from 55-100% POPC and up to 10 mol % PEG-lipid.
In other embodiments, the lipid nanoparticles of the disclosure may
include one or more other lipids including phosphoglycerides,
representative examples of which include phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
phosphatidic acid, palmitoyloleoylphosphatidylcholine,
lyosphosphatidylcholine, lysophosphatidylethanolamine,
dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, di
stearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.
Other compounds lacking in phosphorus, such as sphingolipid and
glycosphingolipid families are useful. Triacylglycerols are also
useful.
Representative nanoparticles of the disclosure have a diameter from
about 10 to about 100 nm. The lower diameter limit is from about 10
to about 15 nm.
The limit size lipid nanoparticles of the disclosure can include
one or more therapeutic and/or diagnostic agents. These agents are
typically contained within the particle core. The nanoparticles of
the disclosure can include a wide variety of therapeutic and/or
diagnostic agents.
Suitable therapeutic agents include chemotherapeutic agents (i.e.,
anti-neoplastic agents), anesthetic agents, beta-adrenaergic
blockers, anti-hypertensive agents, anti-depressant agents,
anti-convulsant agents, anti-emetic agents, antihistamine agents,
anti-arrhytmic agents, and anti-malarial agents.
Representative antineoplastic agents include doxorubicin,
daunorubicin, mitomycin, bleomycin, streptozocin, vinblastine,
vincristine, mechlorethamine, hydrochloride, melphalan,
cyclophosphamide, triethylenethiophosphoramide, carmaustine,
lomustine, semustine, fluorouracil, hydroxyurea, thioguanine,
cytarabine, floxuridine, decarbazine, cisplatin, procarbazine,
vinorelbine, ciprofloxacion, norfloxacin, paclitaxel, docetaxel,
etoposide, bexarotene, teniposide, tretinoin, isotretinoin,
sirolimus, fulvestrant, valrubicin, vindesine, leucovorin,
irinotecan, capecitabine, gemcitabine, mitoxantrone hydrochloride,
oxaliplatin, adriamycin, methotrexate, carboplatin, estramustine,
and pharmaceutically acceptable salts and thereof.
In another embodiment, lipid nanoparticles, are nucleic-acid lipid
nanoparticles.
The term "nucleic acid-lipid nanoparticles" refers to lipid
nanoparticles containing a nucleic acid. The lipid nanoparticles
include one or more cationic lipids, one or more second lipids, and
one or more nucleic acids.
Cationic lipid. The lipid nanoparticles include a cationic lipid.
As used herein, the term "cationic lipid" refers to a lipid that is
cationic or becomes cationic (protonated) as the pH is lowered
below the pK of the ionizable group of the lipid, but is
progressively more neutral at higher pH values. At pH values below
the pK, the lipid is then able to associate with negatively charged
nucleic acids (e.g., oligonucleotides). As used herein, the term
"cationic lipid" includes zwitterionic lipids that assume a
positive charge on pH decrease.
The term "cationic lipid" refers to any of a number of lipid
species which carry a net positive charge at a selective pH, such
as physiological pH. Such lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);
N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA);
N,N-distearyl-N,N-dimethylammonium bromide (DDAB);
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP); 3-(N--(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
(DC-Chol) and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide (DMRIE). Additionally, a number of commercial preparations
of cationic lipids are available which can be used in the present
disclosure. These include, for example, LIPOFECTIN.RTM.
(commercially available cationic liposomes comprising DOTMA and
1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, Grand
Island, N.Y.); LIPOFECTAMINE.RTM. (commercially available cationic
liposomes comprising
N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethy-
lammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and
TRANSFECTAM.RTM. (commercially available cationic lipids comprising
dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from
Promega Corp., Madison, Wis.). The following lipids are cationic
and have a positive charge at below physiological pH: DODAP, DODMA,
DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),
1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).
In one embodiment, the cationic lipid is an amino lipid. Suitable
amino lipids useful in the disclosure include those described in WO
2009/096558, incorporated herein by reference in its entirety.
Representative amino lipids include
1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),
1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),
1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),
1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),
1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),
1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt
(DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride
salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-dioleylamino)-1,2-propanedio (DOAP),
1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-EG-DMA), and
2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane
(DLin-K-DMA).
Suitable amino lipids include those having the formula:
##STR00001##
wherein R.sub.1 and R.sub.2 are either the same or different and
independently optionally substituted C.sub.10-C.sub.24 alkyl,
optionally substituted C.sub.10-C.sub.24 alkenyl, optionally
substituted C.sub.10-C.sub.24 alkynyl, or optionally substituted
C.sub.10-C.sub.24 acyl;
R.sub.3 and R.sub.4 are either the same or different and
independently optionally substituted C.sub.1-C.sub.6 alkyl,
optionally substituted C.sub.2-C.sub.6 alkenyl, or optionally
substituted C.sub.2-C.sub.6 alkynyl or R.sub.3 and R.sub.4 may join
to form an optionally substituted heterocyclic ring of 4 to 6
carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and
oxygen;
R.sub.5 is either absent or present and when present is hydrogen or
C.sub.1-C.sub.6 alkyl;
m, n, and p are either the same or different and independently
either 0 or 1 with the proviso that m, n, and p are not
simultaneously 0;
q is 0, 1, 2, 3, or 4; and
Y and Z are either the same or different and independently O, S, or
NH.
In one embodiment, R.sub.1 and R.sub.2 are each linoleyl, and the
amino lipid is a dilinoleyl amino lipid. In one embodiment, the
amino lipid is a dilinoleyl amino lipid.
A representative useful dilinoleyl amino lipid has the formula:
##STR00002##
wherein n is 0, 1, 2, 3, or 4.
In one embodiment, the cationic lipid is a DLin-K-DMA. In one
embodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA above,
wherein n is 2).
Other suitable cationic lipids include cationic lipids, which carry
a net positive charge at about physiological pH, in addition to
those specifically described above,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);
N-(2,3-dioleyloxy)propyl-N,N--N-triethylammonium chloride (DOTMA);
N,N-distearyl-N,N-dimethylammonium bromide (DDAB);
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP); 1,2-dioleyloxy-3-trimethylaminopropane chloride salt
(DOTAP.Cl);
3.beta.-(N--(N',N'-dimethylaminoethane)carbamoyl)cholesterol
(DC-Chol),
N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethy-
lammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl
carboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane
(DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide (DMRIE). Additionally, a number of commercial preparations
of cationic lipids can be used, such as, e.g., LIPOFECTIN
(including DOTMA and DOPE, available from GIBCO/BRL), and
LIPOFECTAMINE (comprising DOSPA and DOPE, available from
GIBCO/BRL).
The cationic lipid is present in the lipid particle in an amount
from about 30 to about 95 mole percent. In one embodiment, the
cationic lipid is present in the lipid particle in an amount from
about 30 to about 70 mole percent. In one embodiment, the cationic
lipid is present in the lipid particle in an amount from about 40
to about 60 mole percent.
In one embodiment, the lipid particle includes ("consists of") only
of one or more cationic lipids and one or more nucleic acids.
Second lipids. In certain embodiments, the lipid nanoparticles
include one or more second lipids. Suitable second lipids stabilize
the formation of nanoparticles during their formation.
The term "lipid" refers to a group of organic compounds that are
esters of fatty acids and are characterized by being insoluble in
water but soluble in many organic solvents. Lipids are usually
divided in at least three classes: (1) "simple lipids" which
include fats and oils as well as waxes; (2) "compound lipids" which
include phospholipids and glycolipids; and (3) "derived lipids"
such as steroids.
Suitable stabilizing lipids include neutral lipids and anionic
lipids.
Neutral Lipid. The term "neutral lipid" refers to any one of a
number of lipid species that exist in either an uncharged or
neutral zwitterionic form at physiological pH. Representative
neutral lipids include diacylphosphatidylcholines,
diacylphosphatidylethanolamines, ceramides, sphingomyelins,
dihydrosphingomyelins, cephalins, and cerebrosides.
Exemplary lipids include, for example,
distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine
(DOPC), dipalmitoylphosphatidylcholine (DPPC),
dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE) and
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), and
1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE).
In one embodiment, the neutral lipid is
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
Anionic Lipid. The term "anionic lipid" refers to any lipid that is
negatively charged at physiological pH. These lipids
includephosphatidylglycerol, cardiolipin, diacylphosphatidylserine,
diacylphosphatidic acid, N-dodecanoylphosphatidylethanol-amines,
N-succinylphosphatidylethanolamines,
N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,
palmitoyloleyolphosphatidylglycerol (POPG), and other anionic
modifying groups joined to neutral lipids.
Other suitable lipids include glycolipids (e.g.,
monosialoganglioside GM.sub.1). Other suitable second lipids
include sterols, such as cholesterol.
Polyethylene glycol-lipids. In certain embodiments, the second
lipid is a polyethylene glycol-lipid. Suitable polyethylene
glycol-lipids include PEG-modified phosphatidylethanolamine,
PEG-modified phosphatidic acid, PEG-modified ceramides (e.g.,
PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified
diacylglycerols, PEG-modified dialkylglycerols. Representative
polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and
PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is
N-[(methoxy poly(ethylene
glycol).sub.2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine
(PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is
PEG-c-DOMG).
In certain embodiments, the second lipid is present in the lipid
particle in an amount from about 0.5 to about 10 mole percent. In
one embodiment, the second lipid is present in the lipid particle
in an amount from about 1 to about 5 mole percent. In one
embodiment, the second lipid is present in the lipid particle in
about 1 mole percent.
Nucleic Acids. The lipid nanoparticles of the present disclosure
are useful for the systemic or local delivery of nucleic acids. As
described herein, the nucleic acid is incorporated into the lipid
particle during its formation.
As used herein, the term "nucleic acid" is meant to include any
oligonucleotide or polynucleotide. Fragments containing up to 50
nucleotides are generally termed oligonucleotides, and longer
fragments are called polynucleotides. In particular embodiments,
oligonucleotides of the present disclosure are 20-50 nucleotides in
length. In the context of this disclosure, the terms
"polynucleotide" and "oligonucleotide" refer to a polymer or
oligomer of nucleotide or nucleoside monomers consisting of
naturally occurring bases, sugars and intersugar (backbone)
linkages. The terms "polynucleotide" and "oligonucleotide" also
includes polymers or oligomers comprising non-naturally occurring
monomers, or portions thereof, which function similarly. Such
modified or substituted oligonucleotides are often preferred over
native forms because of properties such as, for example, enhanced
cellular uptake and increased stability in the presence of
nucleases. Oligonucleotides are classified as
deoxyribooligonucleotides or ribooligonucleotides. A
deoxyribooligonucleotide consists of a 5-carbon sugar called
deoxyhbose joined covalently to phosphate at the 5' and 3' carbons
of this sugar to form an alternating, unbranched polymer. A
ribooligonucleotide consists of a similar repeating structure where
the 5-carbon sugar is ribose. The nucleic acid that is present in a
lipid particle according to this disclosure includes any form of
nucleic acid that is known. The nucleic acids used herein can be
single-stranded DNA or RNA, or double-stranded DNA or RNA, or
DNA-RNA hybrids. Examples of double-stranded DNA include structural
genes, genes including control and termination regions, and
self-replicating systems such as viral or plasmid DNA. Examples of
double-stranded RNA include siRNA and other RNA interference
reagents. Single-stranded nucleic acids include antisense
oligonucleotides, ribozymes, microRNA, mRNA, and triplex-forming
oligonucleotides.
In one embodiment, the polynucleic acid is an antisense
oligonucleotide. In certain embodiments, the nucleic acid is an
antisense nucleic acid, a ribozyme, tRNA, snRNA, siRNA, shRNA,
ncRNA, miRNA, mRNA, lncRNA, sgRNA, pre-condensed DNA, or an
aptamer.
The term "nucleic acids" also refers to ribonucleotides,
deoxynucleotides, modified ribonucleotides, modified
deoxyribonucleotides, modified phosphate-sugar-backbone
oligonucleotides, other nucleotides, nucleotide analogs, and
combinations thereof, and can be single stranded, double stranded,
or contain portions of both double stranded and single stranded
sequence, as appropriate.
The term "nucleotide", as used herein, generically encompasses the
following terms, which are defined below: nucleotide base,
nucleoside, nucleotide analog, and universal nucleotide.
The term "nucleotide base", as used herein, refers to a substituted
or unsubstituted parent aromatic ring or rings. In some
embodiments, the aromatic ring or rings contain at least one
nitrogen atom. In some embodiments, the nucleotide base is capable
of forming Watson-Crick and/or Hoogsteen hydrogen bonds with an
appropriately complementary nucleotide base. Exemplary nucleotide
bases and analogs thereof include, but are not limited to, purines
such as 2-aminopurine, 2,6-diaminopurine, adenine (A),
ethenoadenine, N6-2-isopentenyladenine (6iA),
N6-2-isopentenyl-2-methylthioadenine (2ms6iA), N6-methyladenine,
guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine
(7mG), 2-thiopyrimidine, 6-thioguanine (6sG) hypoxanthine and
06-methylguanine; 7-deaza-purines such as 7-deazaadenine
(7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines such as
cytosine (C), 5-propynylcytosine, isocytosine, thymine (T),
4-thiothymine (4sT), 5,6-dihydrothymine, O4-methylthymine, uracil
(U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D);
indoles such as nitroindole and 4-methylindole; pyrroles such as
nitropyrrole; nebularine; base (Y); In some embodiments, nucleotide
bases are universal nucleotide bases. Additional exemplary
nucleotide bases can be found in Fasman, 1989, Practical Handbook
of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca
Raton, Fla., and the references cited therein. Further examples of
universal bases can be found for example in Loakes, N. A. R. 2001,
vol 29:2437-2447 and Seela N. A. R. 2000, vol 28:3224-3232.
The term "nucleoside", as used herein, refers to a compound having
a nucleotide base covalently linked to the C-1' carbon of a pentose
sugar. In some embodiments, the linkage is via a heteroaromatic
ring nitrogen. Typical pentose sugars include, but are not limited
to, those pentoses in which one or more of the carbon atoms are
each independently substituted with one or more of the same or
different --R, --OR, --NRR or halogen groups, where each R is
independently hydrogen, (C1-C6) alkyl or (C5-C14) aryl. The pentose
sugar may be saturated or unsaturated. Exemplary pentose sugars and
analogs thereof include, but are not limited to, ribose,
2'-deoxyribose, 2'-(C1-C6)alkoxyribose, 2'-(C5-C14)aryloxyribose,
2',3'-dideoxyribose, 2',3'-didehydroribose, 2'-deoxy-3'-haloribose,
2'-deoxy-3'-fluororibose, 2'-deoxy-3'-chlororibose,
2'-deoxy-3'-aminoribose, 2'-deoxy-3'-(C1-C6)alkylribose,
2'-deoxy-3'-(C1-C6)alkoxyribose and
2'-deoxy-3'-(C5-C14)aryloxyribose. Also see, e.g., 2'-O-methyl,
4'-.alpha.-anomeric nucleotides, 1'-.alpha.-anomeric nucleotides
(Asseline (1991) Nucl. Acids Res. 19:4067-74), 2'-4'- and
3'-4'-linked and other "locked" or "LNA", bicyclic sugar
modifications (WO 98/22489; WO 98/39352; WO 99/14226). "LNA" or
"locked nucleic acid" is a DNA analogue that is conformationally
locked such that the ribose ring is constrained by a methylene
linkage between the 2'-oxygen and the 3'- or 4'-carbon. The
conformation restriction imposed by the linkage often increases
binding affinity for complementary sequences and increases the
thermal stability of such duplexes.
Sugars include modifications at the 2'- or 3'-position such as
methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy,
methoxyethyl, alkoxy, phenoxy, azido, amino, alkylamino, fluoro,
chloro and bromo. Nucleosides and nucleotides include the natural D
configurational isomer (D-form), as well as the L configurational
isomer (L-form) (Beigelman, U.S. Pat. No. 6,251,666; Chu, U.S. Pat.
No. 5,753,789; Shudo, EP0540742; Garbesi (1993) Nucl. Acids Res.
21:4159-65; Fujimori (1990) J. Amer. Chem. Soc. 112:7435; Urata,
(1993) Nucleic Acids Symposium Ser. No. 29:69-70). When the
nucleobase is purine, e.g., A or G, the ribose sugar is attached to
the N9-position of the nucleobase. When the nucleobase is
pyrimidine, e.g., C, T or U, the pentose sugar is attached to the
N1-position of the nucleobase (Kornberg and Baker, (1992) DNA
Replication, 2.sup.nd Ed., Freeman, San Francisco, Calif.).
One or more of the pentose carbons of a nucleoside may be
substituted with a phosphate ester. In some embodiments, the
phosphate ester is attached to the 3'- or 5'-carbon of the pentose.
In some embodiments, the nucleosides are those in which the
nucleotide base is a purine, a 7-deazapurine, a pyrimidine, a
universal nucleotide base, a specific nucleotide base, or an analog
thereof.
The term "nucleotide analog", as used herein, refers to embodiments
in which the pentose sugar and/or the nucleotide base and/or one or
more of the phosphate esters of a nucleoside may be replaced with
its respective analog. In some embodiments, exemplary pentose sugar
analogs are those described above. In some embodiments, the
nucleotide analogs have a nucleotide base analog as described
above. In some embodiments, exemplary phosphate ester analogs
include, but are not limited to, alkylphosphonates,
methylphosphonates, phosphoramidates, phosphotriesters,
phosphorothioates, phosphorodithioates, phosphoroselenoates,
phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates,
phosphoroamidates, boronophosphates, and may include associated
counterions. Other nucleic acid analogs and bases include for
example intercalating nucleic acids (INAs, as described in
Christensen and Pedersen, 2002), and AEGIS bases (Eragen, U.S. Pat.
No. 5,432,272). Additional descriptions of various nucleic acid
analogs can also be found for example in (Beaucage et al.,
Tetrahedron 49(10):1925 (1993) and references therein; Letsinger,
J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem.
81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986);
Sawai et al., Chem. Lett. 805 (1984), Letsinger et al., J. Am.
Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta
26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res.
19:1437 (1991); and U.S. Pat. No. 5,644,048. Other nucleic analogs
comprise phosphorodithioates (Briu et al., J. Am. Chem. Soc.
111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein,
Oligonucleotides and Analogues: A Practical Approach, Oxford
University Press), those with positive backbones (Denpcy et al.,
Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones
(U.S. Pat. Nos. 5,386,023, 5,386,023, 5,637,684, 5,602,240,
5,216,141, and 4,469,863. Kiedrowshi et al., Angew. Chem. Intl. Ed.
English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470
(1988); Letsinger et al., Nucleoside & Nucleotide 13:1597
(194): Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate
Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan
Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett.
4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994);
Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones,
including those described in U.S. Pat. Nos. 5,235,033 and
5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,
"Carbohydrate Modifications in Antisense Research", Ed. Y. S.
Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp
169-176). Several nucleic acid analogs are also described in Rawls,
C & E News Jun. 2, 1997 page 35.
The term "universal nucleotide base" or "universal base", as used
herein, refers to an aromatic ring moiety, which may or may not
contain nitrogen atoms. In some embodiments, a universal base may
be covalently attached to the C-1' carbon of a pentose sugar to
make a universal nucleotide. In some embodiments, a universal
nucleotide base does not hydrogen bond specifically with another
nucleotide base. In some embodiments, a universal nucleotide base
hydrogen bonds with nucleotide base, up to and including all
nucleotide bases in a particular target polynucleotide. In some
embodiments, a nucleotide base may interact with adjacent
nucleotide bases on the same nucleic acid strand by hydrophobic
stacking. Universal nucleotides include, but are not limited to,
deoxy-7-azaindole triphosphate (d7AITP), deoxyisocarbostyril
triphosphate (dICSTP), deoxypropynylisocarbostyril triphosphate
(dPICSTP), deoxymethyl-7-azaindole triphosphate (dM7AITP),
deoxyImPy triphosphate (dImPyTP), deoxyPP triphosphate (dPPTP), or
deoxypropynyl-7-azaindole triphosphate (dP7AITP). Further examples
of such universal bases can be found, inter alia, in Published U.S.
application Ser. No. 10/290,672, and U.S. Pat. No. 6,433,134.
As used herein, the terms "polynucleotide" and "oligonucleotide"
are used interchangeably and mean single-stranded and
double-stranded polymers of nucleotide monomers, including
2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by
internucleotide phosphodiester bond linkages, e.g., 3'-5' and
2'-5', inverted linkages, e.g., 3'-3' and 5'-5', branched
structures, or internucleotide analogs. Polynucleotides have
associated counter ions, such as H+, NH4+, trialkylammonium, Mg2+,
Na+, and the like. A polynucleotide may be composed entirely of
deoxyribonucleotides, entirely of ribonucleotides, or chimeric
mixtures thereof. Polynucleotides may be comprised of
internucleotide, nucleobase and/or sugar analogs. Polynucleotides
typically range in size from a few monomeric units, e.g., 3-40 when
they are more commonly frequently referred to in the art as
oligonucleotides, to several thousands of monomeric nucleotide
units. Unless denoted otherwise, whenever a polynucleotide sequence
is represented, it will be understood that the nucleotides are in
5' to 3' order from left to right and that "A" denotes
deoxyadenosine, "C" denotes deoxycytosine, "G" denotes
deoxyguanosine, and "T" denotes thymidine, unless otherwise
noted.
As used herein, "nucleobase" means those naturally occurring and
those non-naturally occurring heterocyclic moieties commonly known
to those who utilize nucleic acid technology or utilize peptide
nucleic acid technology to thereby generate polymers that can
sequence specifically bind to nucleic acids. Non-limiting examples
of suitable nucleobases include: adenine, cytosine, guanine,
thymine, uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil,
5-methlylcytosine, pseudoisocytosine, 2-thiouracil and
2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine),
N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine),
N9-(7-deaza-8-aza-guanine) and N8-(7-deaza-8-aza-adenine). Other
non-limiting examples of suitable nucleobase include those
nucleobases illustrated in FIGS. 2(A) and 2(B) of Buchardt et al.
(WO92/20702 or WO92/20703).
As used herein, "nucleobase sequence" means any segment, or
aggregate of two or more segments (e.g. the aggregate nucleobase
sequence of two or more oligomer blocks), of a polymer that
comprises nucleobase-containing subunits. Non-limiting examples of
suitable polymers or polymers segments include
oligodeoxynucleotides (e.g. DNA), oligoribonucleotides (e.g. RNA),
peptide nucleic acids (PNA), PNA chimeras, PNA combination
oligomers, nucleic acid analogs and/or nucleic acid mimics.
As used herein, "polynucleobase strand" means a complete single
polymer strand comprising nucleobase subunits. For example, a
single nucleic acid strand of a double stranded nucleic acid is a
polynucleobase strand.
As used herein, "nucleic acid" is a nucleobase sequence-containing
polymer, or polymer segment, having a backbone formed from
nucleotides, or analogs thereof.
Preferred nucleic acids are DNA and RNA.
As used herein, nucleic acids may also refer to "peptide nucleic
acid" or "PNA" means any oligomer or polymer segment (e.g. block
oligomer) comprising two or more PNA subunits (residues), but not
nucleic acid subunits (or analogs thereof), including, but not
limited to, any of the oligomer or polymer segments referred to or
claimed as peptide nucleic acids in U.S. Pat. Nos. 5,539,082,
5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336, 5,773,571,
5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053
and 6,107,470; all of which are herein incorporated by reference.
The term "peptide nucleic acid" or "PNA" shall also apply to any
oligomer or polymer segment comprising two or more subunits of
those nucleic acid mimics described in the following publications:
Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters, 4:
1081-1082 (1994); Petersen et al., Bioorganic & Medicinal
Chemistry Letters, 6: 793-796 (1996); Diderichsen et al., Tett.
Lett. 37: 475-478 (1996); Fujii et al., Bioorg. Med. Chem. Lett. 7:
637-627 (1997); Jordan et al., Bioorg. Med. Chem. Lett. 7: 687-690
(1997); Krotz et al., Tett. Lett. 36: 6941-6944 (1995); Lagriffoul
et al., Bioorg. Med. Chem. Lett. 4: 1081-1082 (1994); Diederichsen,
U., Bioorganic & Medicinal Chemistry Letters, 7: 1743-1746
(1997); Lowe et al., J. Chem. Soc. Perkin Trans. 1, (1997) 1:
539-546; Lowe et J. Chem. Soc. Perkin Trans. 11: 547-554 (1997);
Lowe et al., J. Chem. Soc. Perkin Trans. 11:555-560 (1997); Howarth
et al., J. Org. Chem. 62: 5441-5450 (1997); Altmann, K-H et al.,
Bioorganic & Medicinal Chemistry Letters, 7: 1119-1122 (1997);
Diederichsen, U., Bioorganic & Med. Chem. Lett., 8: 165-168
(1998); Diederichsen et al., Angew. Chem. Int. Ed., 37: 302-305
(1998); Cantin et al., Tett. Lett., 38: 4211-4214 (1997); Ciapetti
et al., Tetrahedron, 53: 1167-1176 (1997); Lagriffoule et al.,
Chem. Eur. J., 3: 912-919 (1997); Kumar et al., Organic Letters
3(9): 1269-1272 (2001); and the Peptide-Based Nucleic Acid Mimics
(PENAMS) of Shah et al. as disclosed in WO96/04000.
Polymer Nanoparticles
The term "polymer nanoparticles" refers to polymer nanoparticles
containing a therapeutic material. Polymer nanoparticles have been
developed using, a wide range of materials including, but not
limited to: synthetic homopolymers such as polyethylene glycol,
polylactide, polyglycolide, poly(lactide-coglycolide),
polyacrylates, polymethacrylates, polycaprolactone,
polyorthoesters, polyanhydrides, polylysine, polyethyleneimine;
synthetic copolymers such as poly(lactide-coglycolide),
poly(lactide)-poly(ethylene glycol),
poly(lactide-co-glycolide)-poly(ethylene glycol),
poly(caprolactone)-poly(ethylene glycol); natural polymers such as
cellulose, chitin, and alginate, as well as polymer-therapeutic
material conjugates.
As used herein, the term "polymer" refers to compounds of usually
high molecular weight built up chiefly or completely from a large
number of similar units bonded together. Such polymers include any
of numerous natural, synthetic and semi-synthetic polymers.
The term "natural polymer" refers to any number of polymer species
derived from nature. Such polymers include, but are not limited to
the polysaccharides, cellulose, chitin, and alginate.
The term "synthetic polymer" refers to any number of synthetic
polymer species not found in Nature. Such synthetic polymers
include, but are not limited to, synthetic homopolymers and
synthetic copolymers.
Synthetic homopolymers include, but are not limited to,
polyethylene glycol, polylactide, polyglycolide, polyacrylates,
polymethacrylates, poly _-caprolactone, polyorthoesters,
polyanhydrides, polylysine, and polyethyleneimine.
"Synthetic copolymer" refers to any number of synthetic polymer
species made up of two or more synthetic homopolymer subunits. Such
synthetic copolymers include, but are not limited to,
poly(lactide-co-glycolide), poly(lactide)-poly(ethylene glycol),
poly(lactide-co-glycolide)-poly(ethylene glycol), and
poly(_-caprolactone)-poly(ethylene glycol).
The term "semi-synthetic polymer" refers to any number of polymers
derived by the chemical or enzymatic treatment of natural polymers.
Such polymers include, but are not limited to, carboxymethyl
cellulose, acetylated carboxymethylcellulose, cyclodextrin,
chitosan and gelatin.
As used herein, the term "polymer conjugate" refers to a compound
prepared by covalently, or non-covalently conjugating one or more
molecular species to a polymer. Such polymer conjugates include,
but are not limited to, polymer-therapeutic material
conjugates.
Polymer-therapeutic material conjugate refers to a polymer
conjugate where one or more of the conjugated molecular species is
a therapeutic material. Such polymer-therapeutic material
conjugates include, but are not limited to, polymer-drug
conjugates.
"Polymer-drug conjugate" refers to any number of polymer species
conjugated to any number of drug species. Such polymer drug
conjugates include, but are not limited to, acetyl
methylcellulose-polyethylene glycol-docetaxol.
As used herein, the term "about" indicates that the associated
value can be modified, unless otherwise indicated, by plus or minus
five percent (+/-5%) and remain within the scope of the embodiments
disclosed.
The following example is included for the purpose of illustrating,
not limiting, the described embodiments.
EXAMPLE
Example 1: Disposable Microfluidic Cartridge for Nanoparticle
Manufacture
In one aspect, the fully integrated disposable microfluidic
cartridge consists of an injection molded carrier; the COC
microfluidic mixer structure; a laser cut clamp piece; three
O-rings; three neodymium magnets and is held together using
self-tapping plastic screws. Such a cartridge is illustrated in
FIGS. 1A-3 and 5A-5H.
The carrier (16 mm.times.67 mm.times.55 mm) is injection molded
from polypropylene. It consists of two female luer slip connectors
(IS0594 compliant); an outlet nozzle (4 mm OD, 2 mm ID.times.4 mm);
3 O-ring grooves above the luer connectors and outlet nozzle (5.3
mm OD.times.0.9 mm); a receptacle for the microfluidic device (52
mm.times.36 mm.times.1.6 mm) and outer wall with a thickness of 1.8
mm and a height of 7 mm.
The clamp (63 mm.times.51 mm.times.1/8 in) is laser cut from
acrylic. There are three holes in the clamp into which neodymium
magnets are press fit and six pilot holes for self-taping
screws.
The cartridge is assembled by placing an O-ring into each of the
three grooves and laying a microfluidic mixer structure on top.
Raised guides along the edge of the inside of the cartridge ensure
that the cartridge falls into place with the ports aligned over the
two inlets and the outlet without separate alignment. The acrylic
clamp with neodymium magnets is then laid on top and held in place
using a series of clips. The assembly is then inverted and secured
together using six self-tapping screws.
The bulk of the microfluidic structure is made by injection molding
COC.
The design of the microfluidic structure is similar to those
pictured in the FIGURES The two microfluidic inlet ports are
connected to channels with a square cross-section and travel to
meet at an angle to form a single channel that enters a serpentine
mixing region including a plurality of turns. The single channel
leaves the mixing chamber and exits the device at an outlet
port.
The flow rate through the Microfluidic Cartridge is 1-40 mL/min,
and the time from the fluids meeting to complete mixing (mixing
speed) is 1-3 ms. The pressure is estimated at 100 psi.
The disposable microfluidic cartridge has been demonstrated to be
essentially as efficient as the existing chip/metal holder. Table 1
below presents data related to forming siRNA-lipid nanoparticles on
an existing metal chip holder and chip configuration ("Chip
Holder+Chip") comparted to a disposable microfluidic (MF) cartridge
in accordance with disclosed embodiments. Comparisons are provided
for nanoparticle size (nm); polydispersity index (PDI); and
encapsulation efficiency for siRNA-Lipid Nanoparticles (siRNA-LNP).
The flow rate for all data is 12 mL/min.
TABLE-US-00001 TABLE 1 Comparison of Microfluidic Formation of
siRNA-Lipid Nanoparticles. Size (nm) PDI Encap. Eff. (%) Chip
Holder + Chip 70.9 0.146 98.1 MF Cartridge 65.3 0.122 97.6
In view of the results presented in Table 1, the disposable MF
cartridge has substantially similar performance to the present
state-of-the art non-disposable metal chip holder and chip. While
the performance between the two microfluidic devices is similar,
the disposable MF cartridge provides the added benefit of being
disposable, conveniently sterilisable, and dramatically reducing
set-up complexity and time.
While illustrative embodiments have been illustrated and described,
it will be appreciated that various changes can be made therein
without departing from the spirit and scope of the invention.
* * * * *
References